Spermatogenesis is a complex process regulated by extratesticular factors through the endocrine system, as well as intratesticular factors secreted from the interstitial compartment and within the seminiferous epithelium. In particular, germ cell-Sertoli cell interactions within the seminiferous epithelium appear to be crucial to the regulation of spermatogenesis. Over the past several years, this laboratory's investigations of paracrine interactions between germ cells and Sertoli cells of the rat have demonstrated that pachytene spermatocytes and round spermatids modulate Sertoli cell secretion of transferrin, sulfated glycoprotein-1, sulfated glycoprotein-2, ceruloplasmin, testins, and novel proteins, S1 and S2. During our investigations of the germ cell proteins which modulate Sertoli cell function, we have identified multiple peaks of germ cell secretagogue activity and mitogenic activity. Most recently, we determined that at least 75% of the germ cell mitogenic activity was attributable to a nerve growth factor-like protein which also induces the secretion of the Sertoli cell protein, S2. However, the germ cell-nerve growth factor-like protein (GC-NGF-L) has several characteristics and properties that differ from nerve growth factor-beta (NGF-beta), suggesting that a testis specific form of this family of proteins is regulating paracrine interactions within the seminiferous epithelium. With the use of an NGF-beta antibody probe which binds to GC-NGF-L, and assays of mitogenic activity, neurite outgrowth and Sertoli cell secretion of protein S2, all of which are characteristic of GC-NGF-L activity, we propose to characterize the GC-NGF-L protein and investigate its role within the rat testis. In this context, a major aim of this research proposal is to purify the GC-NGF-L protein and characterize its properties for comparison with NGF-beta. The purified GC-NGF-L protein will then be used to identify its site of action on Sertoli cells with respect to receptors/binding sites, and the role of GC-NGF-L in modulating Sertoli cell function. These experiments should provide significant new information on the mechanisms involved in paracrine interactions between germ cells and Sertoli cells which participate in the regulation of spermatogenesis.